The Protective Effect And Mechanism Of Salvianolic Acid B Against ANIT-induced Liver Injury In Rats

Objective:In this study,a rat model of cholestatic liver injury was established byα-naphthalene isothiocyanate（ANIT）induction.Meanwhile,the anti-cholestatic liver injury molecular mechanism of Salvianolic acid B（SA-B）was further verified by in vitro cell experiments.Methods:Forty-eight healthy male Sprague-Dawley（SD）rats were randomly divided into four groups:normal group,ANIT model group,low dose group of SA-B(15 mg·kg^-1,i.p.,qd)and high dose group of SA-B(30 mg·kg^-1,i.p.,qd).The two groups treated with SA-B were administrated with SA-B for 7 consecutive days and were given ANIT(75 mg·kg^-1,i.g.)on the 5^th day.Rats in control group were administrated with normal saline（i.p.）for 7consecutive days and on the 5^th day,were given the vehicle（i.g.）.The same treatment was done with the model group,but rats were given ANIT(75 mg·kg^-1,i.g.)on the 5^th day.Rats in each group were administrated with the drug on the 7^th day and sacrificed after fasting for 24 h,and their weight and liver weight were weighed.AST、ALT、ALP、γ-GT、DBIL and TBIL were detected by automatic biochemical analyzer.TBA levels were analyzed with Total Bile acid Assay Kit.Hematoxylin&Eosin（H&E）staining was performed to evaluate the changes of morphology in liver tissue.Real-time PCR was used to test the mRNA levels of Bsep、Mrp3、Oatp2、Cyp3a2、Ugt1a1、IL-1β、IL-6、TGF-β、TNF-αand COX-2 in rats’liver tissues;Western blot analysis was used to determinate the expression of the related signaling proteins in NF-κB/IκB、p38MAPK and JNK MAPK pathways.CCK-8 assay was conducted to determinate the effect of SA-B on L02 cell viability.Cells were seeded in 96-well plates.After 12 h adherent growth,cells were treated with different concentrations of SA-B for 24,48,and 72 hours.Five multiple wells were set for each concentration.Viability was detected by CCK-8.Cells were seeded in 60 mm dishes.Subsequently,adherent cells were exposed to ANIT（200μM）for 24 h and followed by treatment with SA-B（100,200,400μM）for another 24h.L02 cells incubated with culture medium or culture medium with 0.1%DMSO served as control.Real-time PCR was used to test the mRNA levels of BSEP、MRP3、OATP2、CYP3A4、UGT1A1、IL-1β、IL-6、TGF-β、TNF-αand COX-2 in L02 cells;Western blot analysis was used to determinate the expression of the related signaling proteins in NF-κB/IκB、p38 MAPK and JNK MAPK pathways.Results:1.Significant decrease in body weight was observed in ANIT-treated group in comparison to the control group（P<0.05）.This was reversed in SA-B treated group where there was a gradual increase from low-dose group to high-dose group（P<0.05）.However,there was no significant difference in body weight and liver weight between the low-dose and high-dose groups（P>0.05）.2.Significant increase in liver index was observed in ANIT-treated group in comparison to the control group（P<0.05）.This was reversed in SA-B treated group where there was a gradual decrease from low-dose group to high-dose group（P<0.05）.However,there was no significant difference in liver index between the low-dose and high-dose groups（P>0.05）.3.Next,we detected the levels of serum markers of liver functions including AST,ALT, ALP,γ-GT,TBIL,DBIL,TBA.Compared with control group,all seven markers were increased in ANIT group（P<0.05）.And compared with model group,all these indicators showed different degrees of reduction in SA-B-treated groups（P<0.05）.Compared with low-dose group,treatment with SA-B at 30 mg·kg^-1 decreased the level of AST,ALP and TBA（P<0.05）.4.To further evaluate the potential of SA-B in relieving the damage caused by cholestasis,H&E staining was used to monitor histopathological changes in the rat liver. Representative images of liver morphology showed that all rats in control group appeared normal while evident biliary epithelial cell proliferation,hepatocyte necrosis were observed in the liver in model group,leading to inflammatory cell infiltration in the portal areas.Compared with the model group,SA-B treatment ameliorated the degree of ANIT-induced histological damage,especially high-dose group.5.ANIT treatment inhibited the expression of Bsep,Cyp3a2 and Ugt1a1,but increased the mRNA levels of Mrp3 and Oatp2（P<0.05）.Compared with model group,co-treatment with SA-B at 15 mg·kg^-1 up-regulated the expression of these genes（P<0.05）.A higher dose of SA-B exhibited a better up-regulation of gene Mrp3,Oatp2,Cyp3a2 and Ugt1a1（P<0.05）.Compared to the control group,the level of NF-κB in nucleus was significantly increased as well as phosphorylated protein of p38 and JNK（P<0.05）;the levels of NF-κB and IκBαin cytoplasm were significantly decreased（P<0.05）.SA-B treatment could reverse this trend compared with model group（P<0.05）.And the expression of IL-1β,IL-6,TNF-α,TGF-βand COX-2 genes in the model group was significantly higher than the control group（P<0.05）.However,SA-B treatment reversed the levels of these inflammatory mediators’genes,especially at the higher dose（P<0.05）.6.The results showed that SA-B had a little toxic effect on L02 cells at three doses tested in this study.Further,we found that the mRNA levels of BSEP,CYP3A4 and Ugt1A1 were strongly downregulated（P<0.05）and the mRNA levels of MRP3,OATP2 were upregulated（P<0.05）with ANIT stimulation and treatment with SA-B reversed the mRNA levels of BSEP,CYP3A4 and UGT1A1（P<0.05）.Interestingly,mRNA expression of MRP3 and OATP2 in cells treated with SA-B at the three doses were higher when compared to the model cells（P<0.05）.Next,we found that ANIT-treatment significantly increased the level of NF-κB in the nucleus and the level of phosphorylated JNK and p38,decreased the level of NF-κB and IκBαin cytoplasm when compared to the vehicle control（0.1%DMSO group,P<0.05）.Treatment with SA-B inhibited NF- κB translocation into nucleus,suppressed the phosphorylation of JNK and p38（P<0.05） and increased the level of NF-κB and IκBαin cytoplasm（P<0.05）.Simultaneously,we used Real-time PCR to detect the gene expression of inflammatory mediators（IL-1β, IL-6,TGF-β,TNF-α,COX-2）.The results showed that ANIT induced these markers compared with control group（P<0.05）and SA-B reversed the effects of ANIT in a dose dependent manner,and high dose of SA-B showed an obvious reduction（P<0.05）.Conclusion:This study demonstrated that SA-B may act as a potential anti-cholestasis agent which promoted bile acid metabolism because of regulating bile acids transports and metabolic enzymes;which supressed inflammation by inhibiting the NF-κB/IκB,p38 MAPK and JNK MAPK in ANIT-induced cholestatic liver injury.Further studies are needed to evaluate the clinical potential of SA-B as an anti-cholestasis agent.